U.S. Patent Application Ser. No. 61/930,684, filed Jan. 23, 2014, is incorporated herein by reference in its entirety.
Almost all forms of research require the detection and quantification of specific analytes to understand and/or characterize the phenomenon under study. Many types of methods have been applied to this detection ranging from the simple (gravimetric) to the technically complex (quantitative real-time RT-PCR and quantitative atomic force microscopy). The use of spectroscopic methods has come to be relied on heavily in analytical chemistry and the life sciences (Skoog & West, 1972). Analysis of the absorption, scattering, luminescence and fluorescence of light by a solute in solution (or a label on that solute) can enable the characterization of that solute. Spectrometers are utilized to measure the light or radiation altering properties of solutes or their surrogate analytes in solution for this purpose. Spectrophotometers or spectrometers can be multipurpose or designed and built for specific purposes. Multipurpose instruments have a common design mechanism described below while those with specific purpose may have alternative components and potentially simplified formats. Specific purpose instruments are less common but are advantageous for differing reasons including expense, size, data quality, environmental factors, mobility, ease of use, and sample concerns. Often the multipurpose instruments, while providing flexibility, have reduced sensitivity than when compared to the single purpose instruments which have been optimized for a single purpose.
Standard spectrophotometers require that the sample in question is placed in a cuvette directly in the path of a beam of light. The pathlength of standard cuvettes is 1 centimeter and thus typically requires significant volumes of sample for analysis. A sensor is placed in the light path downstream for detection of the light altering properties of the sample. The relationship of the light source, sample, and sensor is important for consistency of measured results. The light altering properties of the sample are typically compared against a reference sample (typically the same solvent without the target solute) to provide data regarding differences in light absorption, transmittance, scatter or emission. These differences are then associated with the solute of interest. Given a relatively pure solute with a known light absorption extinction coefficient, it is possible to determine the concentration and purity of that solute in solution using a spectrophotometer. There is a significant possibility for interference in more complex solutions or even due to the thermal energy supplied by the incident light beam. Spectrophotometers were originally using a single measuring beam (single-beam instrument) and correction for inferences was done by subtraction of a reagent blank as described above. To correct for temperature, light and other interferences a dual-beam approach was developed such that the sample with solute is run side by side with a sample without solute and subtracted in real-time to account for environmental and contaminant impacts. The whole science around spectrophotometric methods is very large and involves a highly versatile and powerful set of related techniques (Skoog & West, 1972).
Specialized spectrophotometers are designed with specific assays in mind. Some require smaller sample volumes (from 0.1 mL to nL), and new instruments have been developed that have eliminated the requirement of a cuvette (e.g., the NANODROP® from ThermoFisher for measuring nucleic acids). Other alternatives use specialized cuvettes that have reduced sample volume by the utilization of mirrored surfaces to redirect the light through alternative paths. Another modification to standard spectrophotometers is the ability to scan multi-well plates and arrays. This enables very high throughput, and standardized tests such as enzyme linked immunosorbent assays (Elisa) (Kennedy, Byrne, Fagain, & Berns, 1990; Lequin, 2005). Other examples include inline systems for analysis of products or cell densities for pharmaceutical and chemical reactors and blood monitors that clamp onto a patient's finger.
Some of the more common uses of spectrophotometers are for the determination of solute concentration in solution. For example, measuring the absorbance of light at 260 nm (A260) can determine the concentration of nucleic acids in solution after extraction from biological samples (Grimsley & Pace, 2003; Powerwave, 2006; Sambrook & Russell, 2001; Willfinger, Mackey, & Chomczynski, 1997). Molecules such as protein, guanidinium salts, and phenol can increase the A260 thus providing an inaccurate reading. These are common contaminants from nucleic acid preparations and need to be removed before accurate measurements of the extracted nucleic acids can be relied upon. These same compounds however absorb at other wavelengths. Specifically, protein has a peak absorbance at 260 nm while phenol and guanidinium salts absorb strongly at 230 nm. By comparing the absorption ratio at two wavelengths it is possible to determine the level of contamination by these compounds. Routinely, A260:280 ratios are used to determine the level of protein contamination in a sample while A260:230 ratios are used to determine the contamination due to either guanidinium salts or phenolic compounds. Further processing can reduce the contamination and this can be further confirmed by repeating the optical measurements. For these applications, the relationship of the light source, sample, and sensor must again be consistent amongst samples to be compared for enable obtaining reliable results.
Colorimetric assays are designed for indirect detection of a substance in solution. The target substance may be a chemical compound, enzyme, or unknown mixture of compounds. Colorimetric assays involve chemical or physical reactions that involve a substrate/enzyme or target/ligand of interest. The result of this chemical reaction is the production of a compound or complex that has altered light/radiation absorbing/emitting properties at a specific wavelength. Ideally the change in light properties is linear in response to the concentration of the specific substrate of interest within a useful range of substrate concentration. Some examples include the use of ferrozine to measure iron atoms, Coomassie blue for protein, and para-nitrophenylphosphate for phosphatase activity (Bradford, 1976; Grimsley & Pace, 2003; Martin, Pallen, Wang, & Graves, 1985; Stookey, 1970). Using a spectrofluorometer, it is also possible to detect the fluorescent emission of light from a fluorescent solute substrate or a fluorescently labeled solute for various purposes. Fluorescent spectroscopy can be linked to enzyme assays, studies of photosynthetic activity, cell enumeration, and various other fluorescence-based assays (Mason, 1993). Fluorescent probes and substrates have expanded the potential for sensitive detection of analytes of interest and generated a broad industry focused on its exploitation (Mason, 1993).
A specialized industry has developed about the use of luminescent assays which are at their core spectroscopy. However, in luminescent assays an enzyme/substrate pair are used to study analytes by generation of light within the sample and using the luminometer (the reading spectrometer) to record the light levels (Vdovenko et al, 2010). Advances in luminescent assays are even able to now produce multiple colors of light to allow multiplexed assays and more complexity within a specific sample to be studied.(Gilbert et al., 2011; Wesierska-Gadek, Gueorguieva, Ranftler, & Zerza-Schnitzhofer, 2005).
In general, as described above, spectrophotometers are large and complex instruments. These instruments are applicable in the chemical or biological laboratories for basic research, clinical diagnostics, biotechnology, chemical, and pharmaceutical industries, military applications, homeland security, and forensics laboratory setting. However, the instruments are too bulky to be mobile as they are often attached directly to a computer to drive the instrument and extract or analyze data. In general a focus on miniaturization has occurred but rather than decrease the instrument size (instrument miniaturization) the focus has been on decreasing sample size (assay miniaturization).
There is a real need for miniaturization of the spectroscopy instrument to reduce its overall cost and allow it to be more generally useful in the laboratory or the field. Ideally this would not come with a reduction in specificity or limit of detection (LOD) for the sample of interest. The single purpose NANODROP spectrophotometer has been able to bridge this gap, although is not easily portable and is a single analyte system.
Miniaturized spectrophotometers have been developed previously. However, they have met with little success and were not accepted broadly as a useful reliable instrument. An early device of Paul Hoogestater (US D237982, 1974) developed a battery operated single channel spectrophotometer for field use. This device presumably used an incandescent bulb and simple photosensor positioned on each side of the 1 cm cuvette sample port with an analog output for absorbance or transmittance. This may have been useful to measure samples like culture density and water sample clarity, but it would not have the accuracy necessary for molecular techniques to measure nucleic acid quality or abundance, nor would it work for fluorescence or luminescence. Several other miniature spectrometers have been pursued having applications with different targets and such do not incorporate many of the functional units described here in one instrument. For instance the spectrometer proposed by Ciaccia et al WO2000014496 A1 utilizes a PCMCIA card attached to a laptop to drive an illumination source and detector. This instrument is tied directly to a computer and thus has less mobility and greater expense associated with the instrument. Jung et al (WO2003073457, 2003) describes an instrument utilizing LED illumination and photodiode detection in a device designed to analyze teeth surfaces. This device does not have a sample holder luminescence or fluorescence capability, or wireless connectivity, and is wired to a computer for data transfer and analysis. Such a device provides little mobility. A spectrometer developed by Crowley et al (WO1998022805) again isn't suitable for measurement of liquid samples in a sample port with defined light path as it demonstrates a probe that would be inserted into a patient's body for direct analysis of targeted tissues. Other known devices attempt to be broad spectrum spectrophotometers. Similarly Cheng-Hao et al (US20130308128) and Zhang et al (U.S. Pat. No. 8,345,226, 2013) have developed broad range spectrophotometers that are mobile, but do not serve the utility in a laboratory setting.
Therefore, what is sought is a portable and adaptable spectrometer with sufficient limits of detection to successfully compete with the more bulky and immobile laboratory grade devices, and which overcomes one or more of the limitations and shortcomings set forth above.